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Microtechnology and integrated microsystems to investigate neuronal networks across scales

Periodic Reporting for period 4 - neuroXscales (Microtechnology and integrated microsystems to investigate neuronal networks across scales)

Reporting period: 2021-04-01 to 2022-09-30

This project constituted an interdisciplinary engineering- and systems neuroscience-driven effort, aimed at a deeper understanding of the behavior of neurons or brain cells across scales. Across scales pertained to the spatial domain - from details of subcellular components through single neurons to entire networks - and the temporal domain - from single action potentials to long-term developmental processes. We used rodent cortical neuron cultures and brain slices, more recently also organoids and human induced pluripotent-stem-cell-derived neurons. The project methodology included a combination of engineering, neuroscience and software/algorithm developments: microelectronics-based high-density microelectrode arrays for recording and stimulation of neurons, patch-clamping directly on the microelectrode chips, high-resolution upright confocal microscopy, genetic methods and the use of voltage sensitive dyes, large-scale data handling strategies, and the development of dedicated data analysis and modeling algorithms.

The project provided new insights into details and fundamentals of neuronal features and information processing. Examples included details of axonal signaling, or high-throughput monitoring of action potentials of a large fraction of neurons in networks over extended time to see developmental effects or effects of disturbances. Fundamental findings (neuron stimulability, extracellular signal characteristics) are also relevant to in-vivo or biomedical applications for improving recording quality, stimulation efficiency and safety. The developed methodology will serve a broad range of applications, such as detailed investigations of the mechanisms of neurodegenerative diseases and applications in personalized medicine or pharmascreening of compounds with different types of electrogenic cells.

- Study, at the same time, details of selected neurons or subcellular components (somas, axons, synapses, dendrites in a network context and the corresponding networks.
- Investigate, in detail, on the neuron/subcellular component side:
(i) biophysical information that can be extracted from extra- and intracellular recordings,
(ii) characteristics and roles of axons and axon initial segments (AIS)
(iii) correlations between modulations in axonal signals and postsynaptic signals, and
(iv) input-output functions of neurons and their input from presynaptic neurons.
- Investigate on the network side:
(i) network architectures/organization and functional connectivity,
(ii) network development, and
(iii) reactions upon perturbations: network plasticity and homeostatic effects.
- Use the obtained measurement results and data sets for modeling on different scales.
We used and further developed high-density microelectrode array (HD-MEA)-technology for deciphering details of neuronal signaling across spatiotemporal scales, from subcellular-compartments through individual neurons to network levels. Moreover, we combined upright confocal microscopy, patch clamp and genetically encoded voltage indicators with HD-MEA technology. We developed new methods to evaluate complex multidimensional neuronal-activity data sets and to include those in neuron compartment models. Finally, we studied human induced pluripotent-stem-cell (hiPSC)-derived neurons and brain organoids.

The main results included:

1. Technology: Development of a dual-mode system for rapid network and axon characterization
We developed a dual-mode high-density microelectrode array, which can simultaneously record in (i) full-frame mode and (ii) high-signal-to-noise mode. We demonstrated the capabilities of this platform with a variety of neuronal preparations at subcellular, cellular and network level. Moreover, we developed reliable analysis tools and an automated analysis pipeline.

2. Science: Elucidation of the prominent role of the axonal initial segment (AIS)
By recording voltages of single neurons in dissociated rat cortical cultures through hundreds of densely packed electrodes, we found that the axon initial segment dominates the measured extracellular-action-potential landscape, and – surprisingly - the soma only contributes to a minor extent. Such basic knowledge about which neuronal compartments contribute to the extracellular voltage landscape is important for interpreting results from all electrical readout schemes. Moreover, the AIS was found to be also the most stimulable compartment of a neuron. This knowledge is crucial for precise stimulation of individual targeted neurons to study single-neuron and neuronal-network characteristics.

3. Science: Network characterization: Synaptic basis of spontaneous spiking
We developed a novel experimental platform and analytical tools that, for the first time, enabled simultaneous excitatory and inhibitory (E/I) synaptic activity readouts in a biological network. We found that postsynaptic spiking coincided with the peaks of brief, network state-dependent increases in the input E/I ratio, which suggested a fine-tuned spiking regime driven by input fluctuations. Moreover, we characterized the organization of connection properties (e.g. strength and spiking rate) in unprecedented detail at single-cell level, which revealed a key role of a few inhibitory hub cells in controlling network spiking.

4. Science and Transfer: Tracking of individual single action potentials along axons
We demonstrated a method to noninvasively and directly record individual APs propagating along millimeter-length axonal arbors with hundreds of microelectrodes at microsecond temporal resolution. We found that cortical axons conduct single APs with high temporal precision and reliability.
In the meantime, also our spinoff MaxWell Biosystems has included an “Axon tracking assay” into their portfolio, as axonal parameters proved to be meaningful in identifying and characterizing certain neurodegenerative diseases (ALS, Parkinson). This assay is now routinely used by many scientists worldwide

5. Science and Transfer: Functional characterization of human-derived and advanced in-vitro models - brain organoids – across scales
We used our HD-MEAs, along with upright confocal microscopy to record from developing cerebral organoids and to characterize their spontaneous neuronal activity across scales. We were able to localize and characterize individual neurons, to infer network characteristics and to even follow axonal action potential along their trajectories. A big benefit in this context was the low noise of our switch-matrix-based HD-MEAs, as the relatively immature human iPSC-derived neurons and organoids featured very low signal amplitudes.
Organoid systems have attracted great interest in pharmaceutical industry, as organoids can be generated from human blood or skin cells, and as patient-derived organoids hold great promise as disease models that faithfully recapitulate 3D arrangements und functionality. Therefore, organoids are increasingly explored for drug testing and functional characterization methods are urgently needed. We will continue this research in follow-up collaboration projects with our spinoff and industry.
EM of neurons on chip, false colors
Confocal image of motorneurons on a chip